Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

Pseurotins comprise a family of structurally related Aspergillal natural products having interesting bioactivity. However, little is known about the biosynthetic steps involved in the formation of their complex chemical features. Systematic deletion of the pseurotin biosynthetic genes in A. fumigatus and in vivo and in vitro characterization of the tailoring enzymes to determine the biosynthetic intermediates, and the gene products responsible for the formation of each intermediate, are described. Thus, the main biosynthetic steps leading to the formation of pseurotin A from the predominant precursor, azaspirene, were elucidated. The study revealed the combinatorial nature of the biosynthesis of the pseurotin family of compounds and the intermediates. Most interestingly, we report the first identification of an epoxidase C-methyltransferase bifunctional fusion protein PsoF which appears to methylate the nascent polyketide backbone carbon atom in trans.

DOI： 10.1002/anie.201404804

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry (RSC)  

Coprinoferrin (CPF), originally isolated from a genetically engineered strain (∆laeA) of mushroom fungus Coprinopsis cinerea, is an acylated tripeptide hydroxamate consisting of tandem aligned N5-hexanoyl-N5-hydroxy-L-ornithine with modifications of N-acetyl and...

DOI： 10.1039/d3ob01850d

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Escherichia coli harboring polyketide synthase (pks+ E. coli) has been suggested to contribute to colorectal cancer development. Physical activity is strongly associated with lower colorectal cancer risks, but its effects on pks+ E. coli remain unclear. The aim of this study was to investigate the association between pks+ E. coli prevalence and physical activity. A cross-sectional study was conducted on 222 Japanese adults (27-79-years-old, 73.9% female). Triaxial accelerometers were used to measure light-intensity physical activity, moderate-to-vigorous intensity physical activity, the physical activity level, step-count, and time spent inactive. Fecal samples collected from participants were used to determine the prevalence of pks+ E. coli. Multivariate logistic regression analysis and restricted cubic spline curves were used to examine the association between pks+ E. coli prevalence and physical activity. The prevalence of pks+ E. coli was 26.6% (59/222 participants). The adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for the highest tertile with reference to the lowest tertile of physical activity variables were as follows: light-intensity physical activity (OR 0.63; 95% CI 0.26-1.5), moderate-to-vigorous intensity physical activity (OR 0.85; 95% CI 0.39-1.87), physical activity level (OR 0.69; 95% CI 0.32-1.51), step-count (OR 0.92; 95% CI 0.42-2.00) and time spent inactive (OR 1.30; 95% CI 0.58-2.93). No significant dose-response relationship was found between all physical activity variables and pks+ E. coli prevalence. Our findings did not suggest that physical activity has beneficial effects on the prevalence of pks+ E. coli. Longitudinal studies targeting a large population are needed to clarify this association.

DOI： 10.1038/s41598-023-47442-9

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Authorship：Lead author,　Last author,　Corresponding author   Language：English   Publishing type：Part of collection (book)   Publisher：Springer Nature Singapore  

DOI： 10.1007/978-981-99-1714-3_6

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The increased incidence of obesity in the global population has increased the risk of several chronic inflammation-related diseases, including non-alcoholic steatohepatitis (NASH)-hepatocellular carcinoma (HCC). The progression from NASH to HCC involves a virus-independent liver carcinogenic mechanism; however, we currently lack effective treatment and prevention strategies. Several reports have suggested that fecal volatile organic compounds (VOCs) are strongly associated with NASH-HCC; therefore, we explored the biomarkers involved in its pathogenesis and progression. Fecal samples collected from control and NASH-HCC model STAM mice were subjected to headspace autosampler gas chromatography-electron ionization-mass spectrometry. Non-target profiling analysis identified diacetyl (2,3-butandione) as a fecal VOC that characterizes STAM mice. Although fecal diacetyl levels were correlated with the HCC in STAM mice, diacetyl is known as a cytotoxic/tissue-damaging compound rather than genotoxic or mutagenic; therefore, we examined the effect of bioactivity associated with NASH progression. We observed that diacetyl induced several pro-inflammatory molecules, including tumor necrosis factor-α, cyclooxygenase-2, monocyte chemoattractant protein-1, and transforming growth factor-β, in mouse macrophage RAW264.7 and Kupffer KPU5 cells. Additionally, we observed that diacetyl induced α-smooth muscle actin, one of the hallmarks of fibrosis, in an ex vivo cultured hepatic section, but not in in vitro hepatic stellate TWNT-1 cells. These results suggest that diacetyl would be a potential biomarker of fecal VOC in STAM mice, and its ability to trigger the macrophage-derived inflammation and fibrosis may partly contribute to NASH-HCC carcinogenesis.

DOI： 10.1038/s41598-023-36091-7

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Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1002/ejoc.202300084

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1002/ejoc.202300084

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Diosgenin is an aglycone of dioscin, a major bioactive steroidal saponin found in plants, including Himalayan Paris (Paris polyphylla), fenugreek (Trigonella foenum-graecum), and yam (Dioscorea spp.). We have previously demonstrated that a species of natural yam, Dioscorea japonica, contains a promising bioactive compound diosgenin, which induces anti-carcinogenic and anti-hypertriacylglycerolemic activities. Here, we found for the first time that Japanese yam (D. japonica) bulbils are richer in diosgenin than the edible tubers (rhizomes) and leaves. LC-MS and imaging-MS analyses revealed that diosgenin accumulated in the peripheral region of D. japonica bulbils. Additionally, we performed RNA-seq analysis of D. japonica, and multiple sequence alignment identified D. japonica CYP90 (DjCYP90), the orthologous gene of CYP90G4 in P. polyphylla, CYP90B50 in T. foenum-graecum, CYP90G6 in Dioscorea zingiberensis, and CYP90G in Dioscorea villosa, which encodes a diosgenin biosynthetic rate-limiting enzyme. The expression levels of DjCYP90 were significantly upregulated in D. japonica bulbils than in its rhizomes and leaves. Since diosgenin is one of the most promising functional food factors executing several favorable bioactivities, D. japonica bulbils rich in diosgenin would be a beneficial natural resource.

DOI： 10.1021/acs.jafc.2c08478

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2-Azahypoxanthine (AHX) was first isolated from the culture broth of the fungus Lepista sordida as a fairy ring-inducing compound. It has since been found that a large number of plants and mushrooms produce AHX endogenously and that AHX has beneficial effects on plant growth. The AHX molecule has an unusual, nitrogen-rich 1,2,3-triazine moiety of unknown biosynthetic origin. Here, we establish the biosynthetic pathway for AHX formation in L. sordida. Our results reveal that the key nitrogen sources that are responsible for the 1,2,3-triazine formation are reactive nitrogen species (RNS), which are derived from nitric oxide (NO) produced by NO synthase (NOS). Furthermore, RNS are also involved in the biochemical conversion of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranosyl 5'-monophosphate (AICAR) to AHX-ribotide (AHXR), suggesting that a novel biosynthetic route that produces AHX exists in the fungus. These findings demonstrate a physiological role for NOS in AHX biosynthesis as well as in biosynthesis of other natural products containing a nitrogen-nitrogen bond.

DOI： 10.1039/d2ob00328g

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Anticancer Research USA Inc.  

DOI： 10.21873/invivo.12746

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Biosynthetic genes are not only responsible for the formation of bioactive substances but also suited for other applications including gene therapy. To test the feasibility of human cells producing antibiotics in situ when provided with a heterologous biosynthetic gene, we focused on cytochrome P450, the class of enzymes important in conferring bioactivity to natural product precursors. We selected Fma-P450 that plays a central role in the fumagillin antimicrobial biosynthesis in Aspergillus fumigatus to examine fungal metabolite production by HeLa cells that express fma-P450 heterologously. Here we show that HeLa cells harboring fma-P450 can biosynthesize 5-hydroxyl-β-trans-bergamoten and cytotoxic 5-epi-demethoxyfumagillol when supplemented with the nontoxic precursor β-trans-bergamotene. While the production level was insufficient to effect cell death, we demonstrate that programming human cells to autogenerate antibiotics by introducing a heterologous biosynthetic gene is feasible.

DOI： 10.1002/cbic.202100645

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Language：Japanese   Publisher：日本健康支援学会  

Language：English   Publishing type：Research paper (scientific journal)  

Escherichia coli containing polyketide synthase in the gut microbiota (pks+ E coli) produce a polyketide-peptide genotoxin, colibactin, and are suspected to play a role in the development of colorectal neoplasia. To clarify the role of pks+ E coli in the early stage of tumorigenesis, we investigated whether the pks status of E coli was associated with the prevalence of colorectal neoplasia. This cross-sectional analysis of data from a prospective cohort in Izu Oshima, Japan included asymptomatic residents aged 40-79 years who underwent screening colonoscopy and provided a stool sample. We identified 543 participants with colorectal neoplasia (22 colorectal cancer and 521 adenoma) as cases and 425 participants with normal colon as controls. The pks status of E coli was assayed using stool DNA and specific primers that detected pks+ E coli. The proportion of pks+ E coli was 32.6% among cases and 30.8% among controls. Compared with those with pks- E coli, the odds ratio (OR) (95% confidence interval) for participants with pks+ E coli was 1.04 (0.77-1.41) after adjusting for potential confounders. No statistically significant associations were observed regardless of tumor site or number of colorectal adenoma lesions. However, stratified analyses revealed increased ORs among participants who consumed cereals over the median intake or vegetables under the median intake. Overall, we found no statistically significant association between pks+ E coli and the prevalence of colorectal adenoma lesions among this Japanese cohort. However, positive associations were suggested under certain intake levels of cereals or vegetables.

DOI： 10.1111/cas.15196

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Language：Japanese   Publisher：東京 : 北隆館  

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Language：English   Publishing type：Research paper (scientific journal)  

BACKGROUND: The Escherichia coli strain that is known to produce the genotoxic secondary metabolite colibactin is linked to colorectal oncogenesis. Therefore, understanding the properties of such colibactin-positive E. coli and the molecular mechanism of oncogenesis by colibactin may provide us with opportunities for early diagnosis or prevention of colorectal oncogenesis. While there have been major advances in the characterization of colibactin-positive E. coli and the toxin it produces, the infection route of the clb + strain remains poorly characterized. RESULTS: We examined infants and their treatments during and post-birth periods to examine potential transmission of colibactin-positive E. coli to infants. Here, analysis of fecal samples of infants over the first month of birth for the presence of a colibactin biosynthetic gene revealed that the bacterium may be transmitted from mother to infant through intimate contacts, such as natural childbirth and breastfeeding, but not through food intake. CONCLUSIONS: Our finding suggests that transmission of colibactin-positive E. coli appears to be occurring at the very early stage of life of the newborn and hints at the possibility of developing early preventive measures against colorectal cancer.

DOI： 10.1186/s12866-021-02292-1

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BACKGROUND: Colibactin-producing Escherichia coli containing polyketide synthase (pks+ E. coli) has been shown to be involved in colorectal cancer (CRC) development through gut microbiota analysis in animal models. Stool status has been associated with potentially adverse gut microbiome profiles from fecal analysis in adults. We examined the association between stool patterns and the prevalence of pks+ E. coli isolated from microbiota in fecal samples of 224 healthy Japanese individuals. RESULTS: Stool patterns were determined through factorial analysis using a previously validated questionnaire that included stool frequency, volume, color, shape, and odor. Factor scores were classified into tertiles. The prevalence of pks+ E. coli was determined by using specific primers for pks+ E. coli in fecal samples. Plasma and fecal fatty acids were measured via gas chromatography-mass spectrometry. The prevalence of pks+ E. coli was 26.8%. Three stool patterns identified by factorial analysis accounted for 70.1% of all patterns seen (factor 1: lower frequency, darker color, and harder shape; factor 2: higher volume and softer shape; and factor 3: darker color and stronger odor). Multivariable-adjusted odds ratios (95% confidence intervals) of the prevalence of pks+ E. coli for the highest versus the lowest third of the factor 1 score was 3.16 (1.38 to 7.24; P for trend = 0.006). This stool pattern exhibited a significant positive correlation with fecal isobutyrate, isovalerate, valerate, and hexanoate but showed a significant negative correlation with plasma eicosenoic acid and α-linoleic acid, as well as fecal propionate and succinate. No other stool patterns were significant. CONCLUSIONS: These results suggest that stool patterns may be useful in the evaluation of the presence of tumorigenic bacteria and fecal fatty acids through self-monitoring of stool status without the requirement for specialist technology or skill. Furthermore, it may provide valuable insight about effective strategies for the early discovery of CRC.

DOI： 10.1186/s12866-021-02255-6

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Colibactin is a polyketide-nonribosomal peptide hybrid secondary metabolite that can form interstrand cross-links in double-stranded DNA. Colibactin-producing Escherichia coli has also been linked to colorectal oncogenesis. Thus, there is a strong interest in understanding the role colibactin may play in oncogenesis. Here, using the high-colibactin-producing wild-type E. coli strain we isolated from a clinical sample with the activity-based fluorescent probe we developed earlier, we were able to identify colibactin 770, which was recently identified and proposed as the complete form of colibactin, along with colibactin 788, 406, 416, 420, and 430 derived from colibactin 770 through structural rearrangements and solvolysis. Furthermore, we were able to trap the degrading mature colibactin species by converting the diketone moiety into quinoxaline in situ in the crude culture extract to form colibactin 860 at milligram scale. This allowed us to determine the stereochemically complex structure of the rearranged form of an intact colibactin, colibactin 788, in detail. Furthermore, our study suggested that we were capturing only a few percent of the actual colibactin produced by the microbe, providing a crude quantitative insight into the inherent instability of this compound. Through the structural assignment of colibactins and their degradative products by the combination of LC-HRMS and NMR spectroscopies, we were able to elucidate further the fate of inherently unstable colibactin, which could help acquire a more complete picture of colibactin metabolism and identify key DNA adducts and biomarkers for diagnosing colorectal cancer.

DOI： 10.1021/jacs.1c01495

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Monocyclic aromatic amines, o-toluidine (o-Tol) and its structural analog o-anisidine (o-Ans), are IARC Group 1 and Group 2A urinary bladder carcinogens, respectively, and are involved in metabolic activation and DNA damage. Our recent study revealed that 2-methyl-N4-(2-methylphenyl) benzene-1,4-diamine (MMBD), a p-semidine-type homodimer of o-Tol, was detected and identified in an in vitro reaction of o-Tol with S9 mix and in vivo urinary samples of o-Tol-exposed rats. Potent mutagenic, genotoxic, and cytotoxic activities were reported with MMBD, suggesting its involvement in urinary bladder carcinogenesis. However, it remains unknown whether o-Ans is converted to active metabolites to induce DNA damage in a similar manner as o-Tol. In this study, we report that a novel o-Ans metabolite, 2-methoxy-N4-(2-methoxyphenyl) benzene-1,4-diamine (MxMxBD), a dimer by head-to-tail binding (p-semidine form), was for the first time identified in o-Ans-exposed rat urine. MxMxBD induced a stronger mutagenicity in N-acetyltransferase overexpressed Salmonella typhimurium strains and potent genotoxicity and cytotoxicity in human bladder carcinoma T24 cells compared with o-Ans. These results suggest that MxMxBD may to some extent contribute toward urinary bladder carcinogenesis. In addition to homodimerization, such as MxMxBD, heterodimerizations were observed when o-Ans was coincubated with o-Tol or aniline (Ani) in in vitro reactions with S9 mix. This study highlights the important consideration of homodimerizations and heterodimerizations of monocyclic aromatic amines, including o-Ans, o-Tol, and Ani, in the evaluation of the combined exposure risk of bladder carcinogenesis.

DOI： 10.1021/acs.chemrestox.0c00536

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Natural products, which can be isolated from living organisms worldwide, have played a pivotal role in drug discovery since ancient times. However, it has become more challenging to identify a structurally novel molecule with promising biological activity for pharmaceutical development, mainly due to the limited methodologies for their acquisition. In this review, we summarize our recent studies that activate the biosynthetic potential of filamentous fungi by genetic engineering to harness the metabolic flow for the efficient production of unprecedented natural products. The recent revolution in genome sequencing technology enables the accumulation of vast amounts of information on biosynthetic genes, the blueprint of the molecular construction. Utilizing the established heterologous expression system, activation of the pathway-specific transcription factor coupled with a knockout strategy, and manipulating the global regulatory gene, the biosynthetic genes were exploited to activate biosynthetic pathways and decipher the encoded enzyme functions. We show that this methodology was beneficial for acquiring fungal treasures for drug discovery. These studies also enabled the investigation of the molecular function of natural products in fungal development.

DOI： 10.1007/s11418-020-01466-x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE RESEARCH  

We have previously reported the identification of CghA, a proposed Diels-Alderase responsible for the formation of the bicyclic octalin core of the fungal secondary metabolite Sch210972. Here we show the crystal structure of the CghA-product complex at a resolution of 2.0 angstrom. Our result provides the second structural determination of eukaryotic Diels-Alderases and adds yet another fold to the family of proteins reported to catalyse [4 + 2] cycloaddition reactions. Site-directed mutagenesis-coupled kinetic characterization and computational analyses allowed us to identify key catalytic residues and propose a possible catalytic mechanism. Most interestingly, we were able to rationally engineer CghA such that the mutant was able to catalyse preferentially the formation of the energetically disfavoured exo adduct. This work expands our knowledge and understanding of the emerging and potentially widespread class of natural enzymes capable of catalysing stereoselective Diels-Alder reactions and paves the way towards developing enzymes potentially useful in various bio/synthetic applications.

DOI： 10.1038/s41929-021-00577-2

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Language：English   Publisher：Symposium on the Chemistry of Natural Products Steering Committee  

DOI： 10.24496/tennenyuki.63.0_121-126

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：National Institute of Infectious Diseases, Japanese Journal of Infectious Diseases Editorial Committee  

We investigated the relationship between colibactin-producing (clb+) Escherichia coli and colorectal adenocarcinoma. In total, 729 E. coli colonies were isolated from tumor and surrounding non-tumor regions in resected specimens from 34 Japanese patients; 450 colonies were from tumor regions and 279 from non-tumor regions. clb+ bacteria were found in tumor regions of 11 patients (11/34, 32.4%) and in non-tumor regions of seven of the 11 (7/34, 20.6%). The prevalence of clb+ isolates was 72.7% (327/450) and 44.1% (123/279) in tumor and non-tumor regions, respectively. All the recovered clb+ isolates belonged to the phylogenetic group B2 and were the most predominant type in tumor regions. Hemolytic (α-hemolysin-positive, hlyA+) and non-hemolytic (α-hemolysin-negative, hlyA-) clb+ isolates were obtained from patient #19; however, the prevalence of hlyA+ clb+ isolates was significantly higher in tumor regions (35/43, 81.4%) than in non-tumor regions (3/19, 15.8%). Moreover, a significantly higher production of N-myristoyl-D-asparagine, a byproduct of colibactin biosynthesis, was observed in hlyA+ clb+ isolates than in hlyA- clb+ isolates. Our results suggest that hlyA+ clb+ E. coli may have a selective advantage in colorectal colonization, consequently playing a role in carcinogenesis. The presence of hlyA+ clb+ bacteria in healthy individuals is a risk marker of colorectal cancer.

DOI： 10.7883/yoken.JJID.2020.066

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Basidiomycetes are known to biosynthesize many biologically interesting compounds, including terpenoids. However, they are notoriously difficult to manipulate. Previously, we identified the gene cluster encoding enzymes responsible for the biosynthesis of lagopodins, cuparene-type sesquiterpenoid quinone natural products in Coprinopsis cinerea. In this study, we attempted to increase the productivity of lagopodin B (1) and related pathway products by overexpressing the terpene cyclase gene cop6 in C. cinerea to determine the details of the complex lagopodin and hitoyol biosynthetic pathway. Random integration of the cop6 into the genome of the ku70-deficient C. cinerea strain resulted in an ~2.4-fold increase in the production of 1. However, integration of cop6 into a highly transcribed position within the chromosome we designated as an expression boost area (EBA) resulted in an ~14-fold greater production of 1. Furthermore, the EBA-integration strain allowed us to isolate a previously undetected product 2, which we determined to be the known compound, hydroxylagopodin B. This finding expanded our understanding of the lagopodin-hitoyol biosynthetic pathway and allowed us to hypothesize a possible mechanism for the biosynthesis of a related homodimeric compound, lagopodin C. Our results demonstrate the potential of targeting EBA to integrate key biosynthetic genes into the genome for enhancing the production of difficult-to-obtain compounds for studying the biosynthesis of complex secondary metabolites in basidiomycetes and other complex eukaryotic organisms.

DOI： 10.1038/s41429-020-0355-9

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The relative contribution of diet to colorectal cancer (CRC) incidence is higher than that for other cancers. Animal models have revealed that Escherichia coli containing polyketide synthase (pks+ E. coli) in the gut participates in CRC development. The purpose of this cross-sectional study was to examine the relationship between dietary intake and the prevalence of pks+ E. coli isolated from the microbiota in faecal samples of 223 healthy Japanese individuals. Dietary intake was assessed using a previously validated brief-type self-administered diet history questionnaire. The prevalence of pks+ E. coli was evaluated using faecal samples collected from participants and specific primers that detected pks+ E. coli. The prevalence of pks+ E. coli was 26.9%. After adjusting for baseline confounders, the prevalence of pks+ E. coli was negatively associated with the intake of green tea (odds ratio [OR], 0.59 [95% confidence interval (CI) 0.30-0.88] per 100 g/1,000 kcal increment) and manganese (OR, 0.43 [95% CI 0.22-0.85] per 1 mg/1,000 kcal increment) and was positively associated with male sex (OR, 2.27 [95% CI 1.05-4.91]). While futher studies are needed to validate these findings, these results provide insight into potential dietary interventions for the prevention of CRC.

DOI： 10.1038/s41598-020-72245-7

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When the microfloral composition deteriorates, it triggers low-level chronic inflammation associated with several lifestyle-related diseases including obesity and diabetic mellitus. Fecal volatile organic compounds (VOCs) have been found to differ in gastrointestinal diseases as well as intestinal infection. In this study, to evaluate a potential association between the pathogenesis of lifestyle-related diseases and VOCs in the intestinal tract, fecal VOCs from obese/diabetic KK-Ay mice (KK) or controls (C57BL/6J mice; BL) fed a normal or high fat diet (NFD or HFD) were investigated using headspace sampler-GC-EI-MS. Principal component analysis (PCA) of fecal VOC profiles clearly separated the experimental groups depending on the mouse lineage (KK vs BL) and the diet type (NFD vs HFD). 16 s rRNA sequencing revealed that the PCA distribution of VOCs was in parallel with the microfloral composition. We identified that some volatile metabolites including n-alkanals (nonanal and octanal), acetone and phenol were significantly increased in the HFD and/or KK groups. Additionally, these volatile metabolites induced proinflammatory activity in the RAW264 murine macrophage cell line indicating these bioactive metabolites might trigger low-level chronic inflammation. These results suggest that proinflammatory VOCs detected in HFD-fed and/or diabetic model mice might be novel noninvasive diagnosis biomarkers for diabetes.

DOI： 10.1038/s41598-020-62541-7

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Introduction: Colibactin is a small genotoxic molecule produced by enteric bacteria, including certain Escherichia coli (E. coli) strains harbored in the human large intestine. This polyketide-peptide genotoxin is considered to contribute to the development of colorectal cancer. The colibactin-producing (clb+) microorganisms possess a 54-kilobase genomic island (clb gene cluster). In the present study, to assess the distribution of the clb gene cluster, genotyping analysis was carried out among E. coli strains randomly chosen from the Japan Collection of Microorganisms, RIKEN BRC, Japan. Findings: The analysis revealed that two of six strains possessed a clb gene cluster. These clb+ strains JCM5263 and JCM5491 induced genotoxicity in in vitro micronucleus (MN) tests using rodent CHO AA8 cells. Since the induction level of MN by JCM5263 was high, a bacterial umu test was carried out with a cell extract of the strain, revealing that the extract had SOS-inducing potency in the umu tester bacterium. Conclusion: These results support the observations that the clb gene cluster is widely distributed in nature and clb+E. coli having genotoxic potencies is not rare among microorganisms.

DOI： 10.1186/s41021-020-00149-z

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Language：Japanese   Publisher：Symposium on the Chemistry of Natural Products Steering Committee  

DOI： 10.24496/tennenyuki.62.0_181-186

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Language：Japanese   Publisher：Symposium on the Chemistry of Natural Products Steering Committee  

DOI： 10.24496/tennenyuki.62.0_391-396

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Language：English   Publishing type：Research paper (scientific journal)  

Biosynthesis of certain fungal polyketide-peptide synthetases involves C-methyltransferase activity that adds one or more S-adenosyl-l-methionine-derived methyl groups to the carbon framework. The previously reported PsoF-MT, the stand-alone C-methyltransferase (MT) from the pseurotin biosynthetic pathway that exists as a domain within a trifunctional didomain enzyme PsoF, was characterized crystallographically and kinetically using mutants with substrate analogs to understand how a trans-acting C-MT works and compare it to known polyketide synthase-associated C-MTs. This study identified key active-site residues involved in catalysis and substrate recognition, which led us to propose the mechanism of C-methylation and substrate specificity determinants in PsoF-MT.

DOI： 10.1021/acs.biochem.9b00702

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While high-colibactin-producing Escherichia coli is thought to be associated with colorectal oncogenesis, this study is complicated part due to an inability to isolate colibactin adequately. Here, we created fluorescent probes activated by ClbP, the colibactin-maturing peptidase, to identify high-colibactin-producing strains. Our probe served as a valuable clinical diagnostic tool that allowed simple high-throughput diagnostic screening of clinical samples. Furthermore, the probe also allowed identification of high-colibactin producers that would help advance our understanding of colibactin biosynthesis.

DOI： 10.1021/acs.orglett.9b01345

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Royal Society of Chemistry (RSC)  

<p>Targeted gene knockout in <italic>Coprinopsis cinerea</italic>, yeast <italic>in vivo</italic> bioconversion and <italic>in vitro</italic> assays elucidated the lagopodin biosynthetic pathway, including a complexity-generating network of oxidation steps.</p>

DOI： 10.1039/c8ob02814a

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Japanese Society of Toxicology  

Colibactin is a polyketide-peptide genotoxin produced by enteric bacteria such as E. coli, and is considered to contribute to the development of colorectal cancer. We previously isolated E. coli strains from Japanese colorectal cancer patients, and in the present study we investigated the genotoxic potency of the colibactin-producing (clb+) E. coli strains that carry the polyketide synthases "pks" gene cluster (pks+) and an isogenic clb- mutant in which the colibactin-producing ability is impaired. Measurement of phosphorylated histone H2AX indicated that DNA double strand breaks were induced in mammalian CHO AA8 cells infected with the clb+ E. coli strains. Induction of DNA damage response (SOS response) by crude extract of the clb+ strains was 1.7 times higher than that of the clb- E. coli in an umu assay with a Salmonella typhimurium TA1535/pSK1002 tester strain. Micronucleus test with CHO AA8 cells revealed that infection with the clb+ strains induced genotoxicity, i.e., the frequencies of micronucleated cells infected with clb+ strain were 4-6 times higher than with the clb- strain. Since the intestinal flora are affected by dietary habits that are strongly associated with ethnicity, these data may contribute to both risk evaluation and prevention of colorectal cancer in the Japanese population.

DOI： 10.2131/jts.44.871

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：Society of Synthetic Organic Chemistry  

Colibactin, a secondary metabolite produced by a commensal Escherichia coli harboring pks island, exhibits a genotoxicity to mammalian cells and progresses to colorectal cancer. Regardless of potential risk for the promotion of cancer by colibactin, which shows neither a biological mechanism of action nor determination of chemical structure. Analysis of a genetic mutant of colibactin-producing E. coli enabled us to provide biosynthetic intermediates and shunt products associated with a partial structure of colibactin. This review covers the recent findings of the biosynthesis and implication of chemical structure of colibactin.

DOI： 10.5059/yukigoseikyokaishi.76.490

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Language：Japanese   Publisher：Symposium on the Chemistry of Natural Products Steering Committee  

DOI： 10.24496/tennenyuki.60.0_37-42

CiNii Research

Language：Japanese   Publisher：公益社団法人 日本薬学会  

冬虫夏草という語を初めて目にし「虫なのか草なのか，一体これは何者だろう？」と興味を抱く人は少なくない．昆虫の亡骸からキノコが生えた奇妙な姿形や，その正体が糸状菌(いわゆるカビ)である事実に驚かされたのは筆者だけではないだろう．冬虫夏草の一種であるサナギタケ<i>Cordyceps</i> <i>militaris</i>は，平板培地においてもキノコを形成するため人工栽培法が確立され，国内でも健康食品として市販されている．その成分の1つがコルジセピン(cordycepin：COR)であり，抗がん，免疫賦活，精力増強など，様々な薬理活性を示すことが知られている．CORはアデノシンの3́位デオキシ体であり，アデノシンとの類似性が故にポリメラーゼによる伸長途中のRNA鎖に取り込まれる．ところが，3́位水酸基が欠如しているため更なる伸長反応が起こらず，細胞増殖が阻害される．一方，CORは生体内のアデニンデアミナーゼ(adenine deaminase：ADA)によって容易に薬物代謝(脱アミノ化)され，活性の減弱化した3́-デオキシイノシン（3́-deoxyinosine：3́dI）を生成する．そのため，かつて実施された白血病治療薬としての臨床試験においては，ADA阻害薬であるペントスタチン(pentostatin：PTN)との併用療法が検討されていた．今回，未解明であったCOR生合成遺伝子が同定され，その独特な生合成戦略が明らかにされたので紹介する．<br>なお，本稿は下記の文献に基づいて，その研究成果を紹介するものである.<br>1) Tuli H. S. <i>et</i> <i>al</i>., <i>Life</i> <i>Sci</i>., <b>93</b>, 863-869(2013)．<br>2) Xia Y. <i>et</i> <i>al</i>., <i>Cell</i> <i>Chem</i>. <i>Biol</i>., <b>24</b>, 1479-1489(2017)．<br>3) Wu P. <i>et</i> <i>al</i>., <i>Cell</i> <i>Chem</i>. <i>Biol</i>., <b>24</b>, 171-181(2017)．

DOI： 10.14894/faruawpsj.54.5_458

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Peroxisomes are well-known organelles that are present in most eukaryotic organisms. Mutant phenotypes caused by the malfunction of peroxisomes have been shown in many fungi. However, these have never been investigated in Agaricomycetes, which include white-rot fungi that degrade wood lignin in nature almost exclusively and play an important role in the global carbon cycle. Based on the results of a forward genetics study to identify mutations causing defects in the ligninolytic activity of the white-rot Agaricomycete Pleurotus ostreatus, we report phenotypes of peal disruptants in P. ostreatus, which are defective in two major features of white-rot Agaricomycetes: lignin biodegradation and mushroom formation. Pex1 disruption was also shown to cause defects in the hyphal growth of P. ostreatus on certain sawdust and minimum media. We also demonstrated that peal is essential for fruiting initiation in the non-wood decaying Agaricomycete Coprinopsis cinerea. However, unlike P. ostreatus, significant defects in hyphal growth on the aforementioned agar medium were not observed in C. cinerea. This result, together with previous a cinerea genetic studies, suggests that the regulation mechanisms for the utilization of carbon sources are altered during the evolution of Agaricomycetes or Agaricales.

DOI： 10.1016/j.fgb.2017.10.002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

During the last decade, we have revealed biosynthetic pathways responsible for the formation of important and chemically complex natural products isolated from various organisms through genetic manipulation. Detailed in vivo and in vitro characterizations enabled elucidation of unexpected mechanisms of secondary metabolite biosynthesis. This personal account focuses on our recent efforts in identifying the genes responsible for the biosynthesis of spirotryprostatin, aspoquinolone, Sch 210972, pyranonigrin, fumagillin and pseurotin. We exploit heterologous reconstitution of biosynthetic pathways of interest in our study. In particular, extensive involvement of oxidation reactions is discussed. Heterologous hosts employed here are Saccharomyces cerevisiae, Aspergillus nidulans and A. niger that can also be used to prepare biosynthetic intermediates and product analogs by engineering the biosynthetic pathways using the knowledge obtained by detailed characterizations of the enzymes. (998 char.)

DOI： 10.1002/tcr.201700015

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

To identify natural products and their associated biosynthetic genes from underutilized, difficult-to-manipulate microbes, chemical screening and bioinformatic analysis were employed to identify secondary metabolites and a potentially associated biosynthetic gene cluster. Subsequently, a heterologous expression system was used to confirm the identity of the gene cluster and the proposed biosynthetic mechanism. This approach successfully identified the curvupallide and spirostaphylotrichin biosynthetic pathways in endophytic fungus Curvularia pallescens and the short-chain pyranonigrin biosynthetic pathway in Aspergillus niger.

DOI： 10.1021/acs.orglett.7b00559

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

The regioselective functionalization of non-activated carbon atoms such as aliphatic halogenation is a major synthetic challenge. A novel multifunctional enzyme catalyzing the geminal dichlorination of a methyl group was discovered in Aspergillus oryzae (Koji mold), an important fungus that is widely used for Asian food fermentation. A biosynthetic pathway encoded on two different chromosomes yields mono- and dichlorinated polyketides (diaporthin derivatives), including the cytotoxic dichlorodiaporthin as the main product. Bioinformatic analyses and functional genetics revealed an unprecedented hybrid enzyme (AoiQ) with two functional domains, one for halogenation and one for O-methylation. AoiQ was successfully reconstituted in vivo and in vitro, unequivocally showing that this FADH(2)-dependent enzyme is uniquely capable of the stepwise gem-dichlorination of a non-activated carbon atom on a freestanding substrate. Genome mining indicated that related hybrid enzymes are encoded in cryptic gene clusters in numerous ecologically relevant fungi.

DOI： 10.1002/anie.201604516

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Language：English   Publisher：MDPI AG  

Varieties of alkaloids are known to be produced by various organisms, including bacteria, fungi and plants, as secondary metabolites that exhibit useful bioactivities. However, understanding of how those metabolites are biosynthesized still remains limited, because most of these compounds are isolated from plants and at a trace level of production. In this review, we focus on recent efforts in identifying the genes responsible for the biosynthesis of those nitrogen-containing natural products and elucidating the mechanisms involved in the biosynthetic processes. The alkaloids discussed in this review are ditryptophenaline (dimeric diketopiperazine alkaloid), saframycin (tetrahydroisoquinoline alkaloid), strictosidine (monoterpene indole alkaloid), ergotamine (ergot alkaloid) and opiates (benzylisoquinoline and morphinan alkaloid). This review also discusses the engineered biosynthesis of these compounds, primarily through heterologous reconstitution of target biosynthetic pathways in suitable hosts, such as Escherichia coli, Saccharomyces cerevisiae and Aspergillus nidulans. Those heterologous biosynthetic systems can be used to confirm the functions of the isolated genes, economically scale up the production of the alkaloids for commercial distributions and engineer the biosynthetic pathways to produce valuable analogs of the alkaloids. In particular, extensive involvement of oxidation reactions catalyzed by oxidoreductases, such as cytochrome P450s, during the secondary metabolite biosynthesis is discussed in details.

DOI： 10.3390/molecules21081078

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN ANTIBIOTICS RESEARCH ASSOC  

To rapidly identify novel natural products and their associated biosynthetic genes from underutilized and genetically difficult-to-manipulate microbes, we developed a method that uses (1) chemical screening to isolate novel microbial secondary metabolites, (2) bioinformatic analyses to identify a potential biosynthetic gene cluster and (3) heterologous expression of the genes in a convenient host to confirm the identity of the gene cluster and the proposed biosynthetic mechanism. The chemical screen was achieved by searching known natural product databases with data from liquid chromatographic and high-resolution mass spectrometric analyses collected on the extract from a target microbe culture. Using this method, we were able to isolate two new meroterpenes, subglutinols C (1) and D (2), from an entomopathogenic filamentous fungus Metarhizium robertsii ARSEF 23. Bioinformatics analysis of the genome allowed us to identify a gene cluster likely to be responsible for the formation of subglutinols. Heterologous expression of three genes from the gene cluster encoding a polyketide synthase, a prenyltransferase and a geranylgeranyl pyrophosphate synthase in Aspergillus nidulans A1145 afforded an alpha-pyrone-fused uncyclized diterpene, the expected intermediate of the subglutinol biosynthesis, thereby confirming the gene cluster to be responsible for the subglutinol biosynthesis. These results indicate the usefulness of our methodology in isolating new natural products and identifying their associated biosynthetic gene cluster from microbes that are not amenable to genetic manipulation. Our method should facilitate the natural product discovery efforts by expediting the identification of new secondary metabolites and their associated biosynthetic genes from a wider source of microbes.

DOI： 10.1038/ja.2016.54

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Language：Japanese   Publisher：Symposium on the Chemistry of Natural Products Steering Committee  

<p>Fumagillin and pseurotin biosynthetic gene clusters are encoded in the genome of Aspergillus fumigatus, located not side by side but together in a single region as an intermingled complex gene cluster. We have uncovered the mechanism of biosynthesis of two specific natural products, fumagillin (1) and pseurotin A (2) through mechanistic analysis of key enzymes in this study. Here we present our studies on (a) the biosynthesis of the core structure of 1, (b) the entire biosynthetic pathway of 2and (c) elucidation of the role played by PsoE (glutathione-S-transferase) and F (flavin-containing monooxygenase–methyltransferase) during the formation of the core structure of 2. </p><p>Compound 1 is a terpene–polyketide hybrid known for its antiangiogenic activity exerted through binding to human methionine aminopeptidase II. 1 has a highly oxygenated structure, and contains a penta-substituted cyclohexane that is generated by oxidative cleavage of the bicyclic sesquiterpene β-trans-bergamotene (3). Until now, the chemical nature, order, and biochemical mechanism of all the oxygenative tailoring reactions has remained enigmatic despite the identification of the biosynthetic gene cluster and the use of targeted gene deletion experiments. The most significant finding from our study is that the P450 monooxygenase, Fma-P450, is shown to catalyze successive hydroxylation, bicyclic ring-opening, and two epoxidations that generate the sesquiterpenoid core skeleton of 1.</p><p>Pseurotins comprise a family of structurally related Aspergillal natural products having interesting bioactivities. However, little is known about the biosynthetic steps involved in the formation of their complex chemical features. We systematically deleted the pseurotin biosynthetic genes in A. fumigatus and performed in vivo and in vitrocharacterizations of the tailoring enzymes to determine the biosynthetic intermediates and the gene products responsible for the formation of each intermediate. This allowed us to elucidate the main biosynthetic steps leading to the formation of 2 from the predominant precursor, azaspirene (12). The study illuminated the combinatorial nature of the pseurotin biosynthetic pathway. Most interestingly, we report the first identification of an epoxidase–methyltransferase bifunctional fusion protein PsoF that carries out C-methylation of the nascent polyketide backbone carbon atom in trans.</p><p>Lastly, we identified a novel mechanism of an enzymatic trans-to-cisisomerization of an olefin that plays a part in generating the observed chemical diversity among the pseurotin-type fungal secondary metabolites. In vitro characterizations of pseurotin biosynthetic enzymes revealed that the glutathione-S-transferase PsoE required a participation of the bifunctional epoxidase–C-methyltransferase PsoF to complete the trans-to-cis isomerization of a pathway intermediate, presynerazol (14). The PsoE–glutathione–presynerazol complex crystal structure indicated that stereospecific glutathione–presynerazol conjugate formation is the principal function of PsoE. Moreover, this study revealed PsoF to have an additional, unexpected oxidative isomerase activity, making it a trifunctional enzyme that is key to the complexity generation in pseurotin biosynthesis. </p>

DOI： 10.24496/tennenyuki.58.0_oral34

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Successful activation of the pyranonigrin biosynthetic gene cluster and gene knockout in Aspergillus niger plus in vivo and in vitro assays led to isolation of six new products, including a spiro cydobutane-containing dimeric compound, which served as the basis for the proposed comprehensive pyranonigrin biosynthetic pathway. Two redox enzymes are key to forming the characteristic fused gamma-pyrone core, and a protease homologue performs the exo-methylene formation.

DOI： 10.1021/acs.orglett.5b02435

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Language：English   Publisher：NATURE PUBLISHING GROUP  

DOI： 10.1038/nchembio.1890

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PubMed

Language：Japanese   Publisher：[東京] : 病態代謝研究会  

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Biochemical studies of purified and dissected fungal polyketide synthase and nonribosomal peptide synthetase (PKS-NRPS) hybrid enzymes involved in biosynthesis of pseurotin and aspyridone indicate that one a-methylation step during polyketide synthesis is a prerequisite and a key checkpoint for chain transfer between PKS and NRPS modules. In the absence of the resulting ?-methyl feature, the completed polyketide intermediate is offloaded as an a-pyrone instead of being aminoacylated by the NRPS domain. These examples illustrate that precisely timed tailoring domain activities play critical roles in the overall programming of the iterative PKS (and NRPS) functions.

DOI： 10.1021/ol503179v

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Polyene macrolactams are a class of microbial metabolites, many of which show potent biological activities with unidentified modes of action. Here we report that 8-deoxyheronamide C, a new 20-membered polyene macrolactam from a marine-derived actinomycete Streptomyces sp., is a unique membrane binder. 8-Deoxyheronamide C showed a characteristic sensitivity profile against fission yeast sterol mutant cells, indicating that the metabolite targets cell membranes. We detected tight physical interaction between heronamides including 8-deoxyheronamide C and heronamide C and saturated hydrocarbon chains in lipid membranes using surface plasmon resonance experiments. We further show that heronamides induced abnormal cell wall morphology in fission yeast probably by perturbing the structure of membrane microdomains. This work will accelerate the biological and medical investigation of polyene macrolactams.

DOI： 10.1021/ja500128u

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Fumagillin (1), a meroterpenoid from Aspergillus fumigatus, is known for its antiangiogenic activity due to binding to human methionine aminopeptidase 2. 1 has a highly oxygenated structure containing a penta-substituted cyclohexane that is generated by oxidative cleavage of the bicyclic sesquiterpene beta-trans-bergamotene. The chemical nature, order, and biochemical mechanism of all the oxygenative tailoring reactions has remained enigmatic despite the identification of the biosynthetic gene cluster and the use of targeted-gene deletion experiments. Here, we report the identification and characterization of three oxygenases from the fumagillin biosynthetic pathway, including a multifunctional cytochrome P450 monooxygenase, a hydroxylating nonheme-iron-dependent dioxygenase, and an ABM family monooxygenase for oxidative cleavage of the polyketide moiety. Most significantly, the P450 monooxygenase is shown to catalyze successive hydroxylation, bicyclic ring-opening, and two epoxidations that generate the sesquiterpenoid core skeleton of 1. We also characterized a truncated polyketide synthase with a ketoreductase function that controls the configuration at C-5 of hydroxylated intermediates.

DOI： 10.1021/ja500881e

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Language：English   Publisher：AMER CHEMICAL SOC  

DOI： 10.1038/nchembio.1366

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Language：Japanese   Publisher：[東京] : 病態代謝研究会  

CiNii Research

Authorship：Lead author   Language：English  

DOI： 10.1038/NCHEMBIO.1366

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Postgenomic analysis revealed that many microorganisms carry numerous secondary metabolite biosynthetic genes on their genome. However, activities of those putative genes are not clearly reflected in the metabolic profile of the microorganisms, especially in fungi. A recent genome mining effort is promising in discovering new natural products. However, many fungi and other organisms are not amenable to molecular genetics manipulations, making the study difficult. Here we report successful engineering of Chaetomium globosum, a known producer of various valuable natural products, that allows its genetic manipulation via targeted homologous recombination. This strain permitted us to abolish transcriptional regulators associated with epigenetic silencing of secondary metabolite biosynthetic pathways, leading to the identification of the products generated by different gene clusters and isolation of novel secondary metabolites. We were able to identify six gene clusters that are responsible for the biosynthesis of 11 natural products previously known to be produced by C. globosum, including one cytochalasan and six azaphilone-type compounds. In addition, we isolated two new compounds, mollipilin A and B, that were only recently identified in a related Chaetomium species. Furthermore, our investigation into the mechanism of biosynthesis of those natural products in C. globosum also led to the discovery of a secondary metabolite, aureonitol, that acts like a transcriptional regulator for the biosynthesis of other secondary metabolites. Similar approaches should facilitate exploration of the untapped potential of fungal biosynthetic capability and identification of various unique biological functions that those secondary metabolites possess.

DOI： 10.1021/ja405128k

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Language：English   Publisher：ELSEVIER SCI LTD  

Echinomycin is an antitumor antibiotic secondary metabolite isolated from streptomycetes, whose core structure is biosynthesized by nonribosomal peptide synthetase (NAPS). The echinomycin biosynthetic pathway was successfully reconstituted in Escherichia coli. NRPS often contains a thioesterase domain at its C terminus for cyclorelease of the elongating peptide chain. Those thioesterase domains were shown to exhibit significant substrate tolerance. More recently, an oxidoreductase Ecm17, which forms the disulfide bridge in triostin A, was characterized. Surprisingly, an unrelated disulfide-forming enzyme GliT for gliotoxin biosynthesis was also able to catalyze the same reaction, providing another example of broad substrate specificity in secondary metabolite biosynthetic enzymes. Those promiscuous catalysts can be a valuable tool in generating diversity in natural products analogs we can produce heterologously.

DOI： 10.1016/j.cbpa.2013.06.022

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Language：English   Publisher：ROYAL SOC CHEMISTRY  

In this article, we review recent successful efforts to engineer biosynthesis of several important fungal natural products through heterologous expression of relevant biosynthetic genes in Saccharomyces cerevisiae. We also describe an innovative method of rapidly cloning fungal polyketide synthase or nonribosomal peptide synthetase genes, which can be 5-20 kb or longer, from a pool of total RNA obtained from the fungus of interest using the technique we termed the "overlap extension PCR-yeast homologous recombination (ExRec)" method. The process concomitantly incorporates the cloned genes into yeast expression vectors for biosynthesis of corresponding polyketide and nonribosomal peptide compounds in our engineered S. cerevisiae strain, allowing detailed chemical characterizations to identify the activities of those previously uncharacterized biosynthetic megaenzymes. Studies reviewed here highlight yeast as a useful and versatile host not only for production of various natural products and mechanistic investigation of biosynthetic enzymes, but also for mining of uncharacterized fungal genomes for novel secondary metabolite biosynthetic pathways.

DOI： 10.1039/c3np70037b

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Language：Japanese   Publisher：Symposium on the Chemistry of Natural Products Steering Committee  

<p> In recent years, many secondary metabolite gene clusters, including polyketide biosynthetic genes, non-ribosomal peptide synthetase genes have been uncovered through fungal genome sequencing. However, several transcriptome analyses revealed that a lot of biosynthetic genes are not activated in conventional culture condition. If we can systematically activate such silent biosynthetic gene clusters with genetic modification, it has a possibility of discovering novel natural products with previously unidentified unique structure and biological activities. In this study, we focused putative transcription factor (TF) genes which locating in the biosynthetic gene cluster. We conducted strong activity promoter-based overexpression of TFs in Aspergillus niger, Chaetomium globosum, Arthroderma otae, Trichophyton equinum, and could successfully biosynthesize secondary metabolites 1-9. These chemical structures were characterized based on MS and NMR spectroscopic analysis. The comprehensive relationship between gene clusters and natural products has allowed us to provide insight into their biosynthetic pathway.</p>

DOI： 10.24496/tennenyuki.55.0_oral35

CiNii Research

Language：Japanese   Publisher：Symposium on the Chemistry of Natural Products Steering Committee  

<p>Cell membrane is one of the most challenging research subjects, partly because lipid functions at a molecular level are largely unknown. We are conducting chemical genetics approaches using compounds targeting lipids to elucidate the structure and function of cell membranes. Here we report that heronamides, polyene macrolactams from a marine-derived Streptomces sp., are a unique class of membrane binders. </p><p>We have constructed a screening system to find natural products targeting membrane lipids, using fission yeast as a model organism. During the course of the screening, we isolated heronamides, polyene macrolactams from a marine-derived Streptomyces sp. NSU893. The chemical structures of heronamides including 8-deoxyheronamide C (8-dHC, 1), a new analog, were determined by chemical conversion and spectroscopic analysis. Among heronamides, 8-dHC (1) exhibited unique cell growth inhibition pattern that was typical for membrane binders. To demonstrate the affinity between heronamides and lipid membrane, we examined surface plasmon resonance (SPR) analysis. The sensorgrams indicated that 8-dHC (1) and heronamide C (2) strongly bound lipid membranes consisting of lipids with saturated acyl/alkyl chains. In microscopic analysis, drastic morphological changes were observed when fission yeast cells were exposed to heronamides. Interestingly, this phenomenon was critically similar to that reported for a mutant of sphingolipid metabolism. These results suggest that heronamides target cell membrane probably by binding to membrane lipids, such as sphingolipids.</p><p>In conclusion, we isolated heronamides and revealed that they bound to lipid membranes consisting of saturated acyl/alkyl chains and caused a drastic morphological change in fission yeast cells. Detailed analysis of their modes of action is underway.</p>

DOI： 10.24496/tennenyuki.55.0_oral20

CiNii Research

Publishing type：Research paper (scientific journal)   Publisher：Wiley  

DOI： 10.1002/chin.201247204

Language：Japanese   Publisher：Symposium on the chemistry of natural products  

The article describes the detailed mechanism of the formation of the spiro-carbon moiety in an anti-mitotic fungal nonribosomal peptide (NRP), spirotryprostatin A, mediated by a key enzyme, FAD-dependent monooxygenase (FMO). The presence of spiro-carbon moieties is prevalent among natural products, yet the detailed mechanism of their biosynthesis is poorly characterized. Those spiro-centers remain to be a significant challenge to synthetic chemistry as well. Thus, we sought to investigate how a sterically constrained chiral spiro-carbon could be synthesized enzymatically. We focused on spirotryprostatins because of their useful biological activity and complex chemical structure involving a spiro-carbon moiety. Since similar structural motif is also found in other indole alkaloid natural products, we felt that our findings here will have a broad impact on our understanding of the mechanism of complex natural product biosynthesis. What is frequently the major obstacle in conducting detailed mechanistic studies of enzymes involved in complex natural product biosynthesis is the lack of adequate supply of substrates and reference materials. This was true for studying the spiro-carbon formation in spirotryprostatins. To resolve this problem, we successfully carried out metabolic engineering of yeast for heterologous production of tryprostatins (substrates for spirotryprostatins formation) and other compounds involved in the biosynthesis of tryprostatins and spirotryprostatins using our engineered strain of Saccharomyces cerevisiae, SCKW5. Using the compounds obtained from yeast, we characterized the activity of an FMO, Afua_12060, involved in the fumiquinazoline biosynthesis and confirmed that Afua_12060 was responsible for an poxidation-driven semipinacol-type rearrangement of tryprostatin A that set up the compound for the ensuing formation of spirotryprostatin A. Comparison of our finding with the proposed biosynthetic mechanism of related fumiquinazoline and notoamide natural products revealed that FMO-catalyzed epoxidation of unactivated indole plays a key role in the formation of various complex chemical structures (namely spiro-carbon and imidazoindolone ring systems). However, what was most intriguing about our finding was the discovery of an unusual crosstalk between two biosynthetic pathways (fumiquinazoline and fumitremorgin biosynthetic pathways) that resulted in the formation of new compounds with interesting chemical features (spirotryprostatins).

DOI： 10.24496/tennenyuki.54.0_79

CiNii Books

Authorship：Lead author   Language：English  

CiNii Research

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Tumescenamide C, a new cyclic lipodepsipeptide, was isolated from a culture broth of an actinomycete Streptomyces sp. KUSC_F05. Tumescenamide C was a congener of tumescenamides A and B, representing a sixteen-membered ring system, consisting of two proteinogenic and three non-proteinogenic amino acids, to which a methyl-branched fatty acid was attached. The planar structure was determined by spectroscopic analysis, while its absolute stereochemistry was determined by chemical degradation and asymmetric synthesis. Tumescenamide C exhibited antimicrobial activity with high selectivity against Streptomyces species. (c) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.tet.2012.04.075

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN ANTIBIOTICS RESEARCH ASSOC  

DOI： 10.1038/ja.2012.34

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

Fungal genome sequencing has revealed many genes coding for biosynthetic enzymes, including polyketide synthases and nonribosomal peptide synthetases. However, characterizing these enzymes and identifying the compounds they synthesize remains a challenge, whether the genes are expressed in their original hosts or in more tractable heterologous hosts, such as yeast. Here, we developed a streamlined method for isolating biosynthetic genes from fungal sources and producing bioactive molecules in an engineered Saccharomyces cerevisiae host strain. We used overlap extension PCR and yeast homologous recombination to clone desired fungal polyketide synthase or a nonribosomal peptide synthetase genes (520 kb) into a yeast expression vector quickly and efficiently. This approach was used successfully to clone five polyketide synthases and one nonribosomal peptide synthetase, from various fungal species. Subsequent detailed chemical characterizations of the resulting natural products identified six polyketide and two nonribosomal peptide products, one of which was a new compound. Our system should facilitate investigating uncharacterized fungal biosynthetic genes, identifying novel natural products, and rationally engineering biosynthetic pathways for the production of enzyme analogues possessing modified bioactivity.

DOI： 10.1002/cbic.201100798

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Language：Japanese   Publisher：Symposium on the Chemistry of Natural Products Steering Committee  

The article describes the detailed mechanism of the formation of the spiro-carbon moiety in an anti-mitotic fungal nonribosomal peptide (NRP), spirotryprostatin A, mediated by a key enzyme, FAD-dependent monooxygenase (FMO). The presence of spiro-carbon moieties is prevalent among natural products, yet the detailed mechanism of their biosynthesis is poorly characterized. Those spiro-centers remain to be a significant challenge to synthetic chemistry as well. Thus, we sought to investigate how a sterically constrained chiral spiro-carbon could be synthesized enzymatically. We focused on spirotryprostatins because of their useful biological activity and complex chemical structure involving a spiro-carbon moiety. Since similar structural motif is also found in other indole alkaloid natural products, we felt that our findings here will have a broad impact on our understanding of the mechanism of complex natural product biosynthesis. What is frequently the major obstacle in conducting detailed mechanistic studies of enzymes involved in complex natural product biosynthesis is the lack of adequate supply of substrates and reference materials. This was true for studying the spiro-carbon formation in spirotryprostatins. To resolve this problem, we successfully carried out metabolic engineering of yeast for heterologous production of tryprostatins (substrates for spirotryprostatins formation) and other compounds involved in the biosynthesis of tryprostatins and spirotryprostatins using our engineered strain of Saccharomyces cerevisiae, SCKW5. Using the compounds obtained from yeast, we characterized the activity of an FMO, Afua_12060, involved in the fumiquinazoline biosynthesis and confirmed that Afua_12060 was responsible for an poxidation-driven semipinacol-type rearrangement of tryprostatin A that set up the compound for the ensuing formation of spirotryprostatin A. Comparison of our finding with the proposed biosynthetic mechanism of related fumiquinazoline and notoamide natural products revealed that FMO-catalyzed epoxidation of unactivated indole plays a key role in the formation of various complex chemical structures (namely spiro-carbon and imidazoindolone ring systems). However, what was most intriguing about our finding was the discovery of an unusual crosstalk between two biosynthetic pathways (fumiquinazoline and fumitremorgin biosynthetic pathways) that resulted in the formation of new compounds with interesting chemical features (spirotryprostatins).

DOI： 10.24496/tennenyuki.54.0_79

CiNii Research

Language：Japanese   Publisher：Japan Society for Bioscience, Biotechnology, and Agrochemistry  

DOI： 10.1271/kagakutoseibutsu.50.163

CiNii Books

CiNii Research

Language：Japanese  

CiNii Books

CiNii Research

Language：Japanese   Publisher：Symposium on the Chemistry of Natural Products Steering Committee  

Microbes are a rich source of bioactive metabolites; they represent a wide variety of chemical structures and specific biological activities. In our continuing search for bioactive metabolites from microbes, a culture broth of an actinomycete strain of the genus Streptomyces, from which several bioactive secondary metabolites were isolated, was found to contain a novel antimicrobial lipodepsipeptide, designated pentadepsin A (1). Herein, we report the isolation, structure elucidation and biological activity of pentadepsin A. The molecular formula of 1 was established as C37H57N508 on the basis of HR-FABMS and NMR spectral data. The planar structure of 1 was elucidated by 2D NMR analysis; it represented a sixteen-membered ring system, consisting of five amino acids, to which a unique acyl group 2,4-dimethyl heptanoic acid (Dmh) was linked through an amide bond. The absolute stereochemistry of amino acid residues was determined by advanced Marfey's method, while that of Dmh residue was partially elucidated by PGME method. For the full elucidation of the side chain structure, (2S,4R)-Dmh and (2S,4S)-Dmh were synthesized via Evans alkylation. Spectroscopic comparison of Dmh obtained from 1 with the synthesized ones revealed that the side chain of 1 is (2S,4S)-2,4-dimethylheptanoic acid. Pentadepsin A selectively inhibited cell growth of Streptomyces species, while exhibited no growth inhibition against other bacteria tested and no cytotoxic effect mammalian cells. Detailed analysis of the mechanism of action of pentadepsin A is underway.

DOI： 10.24496/tennenyuki.53.0_397

CiNii Books

CiNii Research

Language：English   Publisher：Pediatric Blood and Cancer  

DOI： 10.1002/pbc.22478

Web of Science

Scopus

PubMed

Language：Japanese   Publisher：Symposium on the Chemistry of Natural Products Steering Committee  

Transforming growth factor-β (TGF-β) facilitates tumor growith and metastasis in advanced cancer. Discovery of novel TGF-β signaling inhibitors may open a new strategy for treatment of cancer patients. However, few clinically-promising drugs that block the TGF-β signaling pathway have been reported. Therefore, we are investigating novel TGF-β signaling inhibitors from natural sources, including microbial metabolites. For the screening assay, we established a luciferase reporter cell line from mink lung epithelial cells, Mv1Lu, to monitor the TGF-β-induced signal. Among over 2000 microbial extracts screened, an extract from an actinomycete Streptomyces sp. was found to inhibit the luciferase activity without cytotoxicity. Three litters of the culture broth were extracted with n-BuOH, filtered and concentrated. An active substance, named tryptopeptin A, was obtained by using silica gel chromatography followed by reverse-phase HPLC, as a colorless solid (3.6mg, 0.023%). Detailed MS and NME spectroscopic analyses revealed that tryptopeptin A was a novel acylated tripeptede, somprising a isovaleric acid, an N-methylvaline, a threonine and a C-terminally modified tryptophan in which carboxylic acid was replaced with an α,β-epoxyketone gropu. The absolute stereochemistries of tryptopeptin A were determined by Marfey's method and a synthetic approach: the physicochemical properties of the synthesized compound were idintical with those of the natural product. Tryptopeptin A potently inhibited TGF-β-dependent luciferase activity without cytotoxicity, suggesting the presence of specific cellular target molecule(s). Structure-activity relationships and modes of action of tryptopeptin A will also be discussed.

DOI： 10.24496/tennenyuki.52.0_397

CiNii Research

Web of Science

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Symbiospirols A, B, and C, long carbon-chain compounds with a molecular weight of 1207 were isolated from the cultured symbiotic dinoflagellate Symbiodinium sp. Their planar structures and partial relative stereochemistries were elucidated based on 1D and 2D NMR spectra and a degradation reaction. Symbiospirols consisted of a C67-linear chain with a beta,beta&apos;-dihydroxyl ketone moiety, eight hydroxyl groups, one tetrahydropyran ring, and two 1,6-dioxaspiro[4,4]nonane rings. Symbiospirol A had an inhibitory effect against L-phosphatidylserine-induced PKC activation.

DOI： 10.1021/ol900299x

Web of Science

Scopus

PubMed

Language：Japanese   Publisher：Symposium on the chemistry of natural products  

Dinoflagellates are widely known as the rich source of biologically active and structural unique secondary metabolites. Among them, large polyols and polyethers composed of a long carbon backbone that is highly functionalized by oxygen, as we say "super-carbon-chain compounds (SCCs)", are some of the most unusual compounds. In our quest for SCCs from marine dinoflagellates, we isolated a novel spiroketal compound, symbiospirol A, from Symbiodinium sp. that was the same strain which produced symbiodinolide. Its planar structure and partial relative stereochemistries were elucidated based on 1D and 2D-NMR and degradation reaction. Symbiospirol A was shown to inhibit PKC activation induced by phosphatidylserine. Symbiodinolide is the typical SCC with a molecular weight of 2,860, which we isolated from the dinoflagellate Symbiodinium sp. The chemical degradation reaction using the 2nd-generation Hoveyda-Grubbs catalyst was employed for the stereochemical assignment at 62-membered macrolactone of symbiodinolide. The absolute configurations at several chiral centers were determined by spectroscopic analysis of MTPA diesters. Further chemical degradation studies using the 2nd-generation Grubbs' catalyst have performed new reaction. In addition, the complete stereochemical assignment of symbiodinolide is in progress. In our continuing search for novel bioactive metabolites from symbiotic marine dinoflagellates, we found that the water layer from the symbiotic dinoflagellate with jellyfish Mastigias papua displayed considerable activity against the expression of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVEC). Further purification of the water layer guided by the bioassay and mass measurement led to the isolation of an unknown bioactive SCC, which we named symbiopolyol, possessing the molecular weight of 1,220. Studies on the isolation, structure elucidation, and biological activities of symbiopolyol will be described.

DOI： 10.24496/tennenyuki.50.0_35

CiNii Books

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

The structure of zamamistatin, an antibacterial dibromotyrosine derivative, has been revised. Its structure was not exo- or endo-type dimer 1 or 2 which were described previously, but was identical to a dibromophenylpyruvic acid derivative, aeroplysinin-1 (3). (c) 2008 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.tetlet.2008.06.125

Web of Science

Scopus

Language：Japanese   Publisher：Symposium on the Chemistry of Natural Products Steering Committee  

Dinoflagellates are widely known as the rich source of biologically active and structural unique secondary metabolites. Among them, large polyols and polyethers composed of a long carbon backbone that is highly functionalized by oxygen, as we say "super-carbon-chain compounds (SCCs)", are some of the most unusual compounds. In our quest for SCCs from marine dinoflagellates, we isolated a novel spiroketal compound, symbiospirol A, from Symbiodinium sp. that was the same strain which produced symbiodinolide. Its planar structure and partial relative stereochemistries were elucidated based on 1D and 2D-NMR and degradation reaction. Symbiospirol A was shown to inhibit PKC activation induced by phosphatidylserine. Symbiodinolide is the typical SCC with a molecular weight of 2,860, which we isolated from the dinoflagellate Symbiodinium sp. The chemical degradation reaction using the 2nd-generation Hoveyda-Grubbs catalyst was employed for the stereochemical assignment at 62-membered macrolactone of symbiodinolide. The absolute configurations at several chiral centers were determined by spectroscopic analysis of MTPA diesters. Further chemical degradation studies using the 2nd-generation Grubbs' catalyst have performed new reaction. In addition, the complete stereochemical assignment of symbiodinolide is in progress. In our continuing search for novel bioactive metabolites from symbiotic marine dinoflagellates, we found that the water layer from the symbiotic dinoflagellate with jellyfish Mastigias papua displayed considerable activity against the expression of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVEC). Further purification of the water layer guided by the bioassay and mass measurement led to the isolation of an unknown bioactive SCC, which we named symbiopolyol, possessing the molecular weight of 1,220. Studies on the isolation, structure elucidation, and biological activities of symbiopolyol will be described.

DOI： 10.24496/tennenyuki.50.0_35

CiNii Research

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

Language：Japanese  

J-GLOBAL

Language：Japanese   Publisher：(一社)日本生薬学会  

J-GLOBAL

Language：Japanese   Publisher：日本細菌学会  

Language：Japanese   Publisher：日本細菌学会  

J-GLOBAL

Language：Japanese   Publisher：日本細菌学会  

Language：Japanese  

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

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J-GLOBAL

Language：Japanese   Publisher：日本癌学会  

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

J-GLOBAL

Language：Japanese  

J-GLOBAL

J-GLOBAL

J-GLOBAL

Language：Japanese  

J-GLOBAL

Language：Japanese  

J-GLOBAL

J-GLOBAL

Language：Japanese   Publisher：Symposium on the chemistry of natural products  

Transforming growth factor-β (TGF-β) facilitates tumor growith and metastasis in advanced cancer. Discovery of novel TGF-β signaling inhibitors may open a new strategy for treatment of cancer patients. However, few clinically-promising drugs that block the TGF-β signaling pathway have been reported. Therefore, we are investigating novel TGF-β signaling inhibitors from natural sources, including microbial metabolites. For the screening assay, we established a luciferase reporter cell line from mink lung epithelial cells, Mv1Lu, to monitor the TGF-β-induced signal. Among over 2000 microbial extracts screened, an extract from an actinomycete Streptomyces sp. was found to inhibit the luciferase activity without cytotoxicity. Three litters of the culture broth were extracted with n-BuOH, filtered and concentrated. An active substance, named tryptopeptin A, was obtained by using silica gel chromatography followed by reverse-phase HPLC, as a colorless solid (3.6mg, 0.023%). Detailed MS and NME spectroscopic analyses revealed that tryptopeptin A was a novel acylated tripeptede, somprising a isovaleric acid, an N-methylvaline, a threonine and a C-terminally modified tryptophan in which carboxylic acid was replaced with an α,β-epoxyketone gropu. The absolute stereochemistries of tryptopeptin A were determined by Marfey's method and a synthetic approach: the physicochemical properties of the synthesized compound were idintical with those of the natural product. Tryptopeptin A potently inhibited TGF-β-dependent luciferase activity without cytotoxicity, suggesting the presence of specific cellular target molecule(s). Structure-activity relationships and modes of action of tryptopeptin A will also be discussed.

DOI： 10.24496/tennenyuki.52.0_397

CiNii Books

J-GLOBAL

Language：Japanese  

J-GLOBAL